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Introduction

Instructions are provided below for silver staining Novex® Gels using the SilverQuest™ Silver Staining Kit and the SilverXpress® Silver Staining Kit. If you are using any other silver staining kit, follow the manufacturer’s recommendations.

SilverQuest™ Microwave Silver Staining Protocol

The Fast Staining protocol (using a microwave oven) for silver staining Novex® Gels using SilverQuest™ Silver Staining Kit is described below. For the Basic Protocol and more details on the staining procedure, refer to the SilverQuest™ Silver Staining Kit Manual (IM-6070). This manual is available on our Web site at www.invitrogen.com or contact Technical Service. For use with an 8 x 8 cm Novex® Gel, 1.0 mm thick. Use 100 ml of each solution per gel.

Note: You may have to optimize the staining protocol, if the dimensions of your gel are not the same as mentioned above.

Caution: Use caution while performing the Fast Staining Protocol using a microwave oven. Do not overheat the staining solutions.

After electrophoresis, place the gel in a clean microwaveable staining tray of the appropriate size. Rinse the gel briefly with ultrapure water.

Place the gel in 100 ml of fixative and microwave at high power (700 watts) for 30 seconds. Remove the gel from the microwave and gently agitate it for 5 minutes at room temperature. Decant the fixative.

Wash the gel with 100 ml of 30% ethanol in a microwave at high power for 30 seconds. Remove the gel from the microwave and gently agitate it for 5 minutes at room temperature on a rotary shaker. Decant the ethanol.

Add 100 ml of Sensitizing solution to the washed gel. Microwave at high power for 30 seconds. Remove the gel from the microwave and place it on a rotary shaker for 2 minutes at room temperature. Decant the Sensitizing solution.

Wash the gel twice in 100 ml ultrapure water. Microwave at high power for 30 seconds. At each wash step, remove the gel from the microwave and gently agitate it for 2 minutes at room temperature.

Place the gel in 100 ml of Staining solution. Microwave at high power for 30 seconds. Remove the gel from the microwave and gently agitate it for 5 minutes at room temperature.

Decant the Staining solution and wash the gel with 100 ml of ultrapure water for 20-60 seconds. Do not wash the gel for more than a minute.

Place the gel in 100 ml of Developing solution and incubate for 5 minutes at room temperature with gentle agitation on a rotary shaker. Do not microwave.

Once the desired band intensity is achieved, immediately add 10 ml of Stopper directly to the gel still immersed in Developing solution and gently agitate the gel for 10 minutes. The color changes from pink to clear indicating that the end of development.

Wash the gel with 100 ml of ultrapure water for 10 minutes. If you need to destain the gel for mass spectrometry analysis, see the SilverQuest™ Silver Staining Kit Manual (IM-6070).

Preparing Solutions for SilverXpress® Silver Staining

Prepare the reagents as described below. If you are staining two gels, double the reagent volumes. Note: The final volumes of solutions containing methanol and water reflect a volume shrinkage which occurs when these two reagents are mixed. Do not adjust volumes of components or final volume.